Therapies involving the genetic modification of stem cells hold substantial promise for the treatment of blood- and immunesystem disorders. They also face daunting obstacles limiting their feasibility and efficiency, related in part to poor target cell access and compromised stem cell survival in culture. We have recently made a series of novel observations that demonstrate the prolonged intracellular retention of HIV-derived lentivector particles by hematopoietic "carrier" cells and their capacity for secondary transduction events. The release of intact particles in murine transplantation studies led to preferential particle dissemination to host hematopoietic organs. These findings reveal a unique therapeutic opportunity to combine particle capture and active carrier cell homing for the delivery of lentivector to the marrow microenvironment. We hypothesize that lentivector particle capture and release by hematopoietic "carrier" cells, when combined with established homing mechanisms, can be exploited and optimized for the genetic modification of bone marrow stem cells in situ. We will systematically test this hypothesis in three specific aims. In a murine transplantation model with a stem cell defect we will ascertain the ability of carrier cells to phenotypically correct long-lived stem cells by in situ particle delivery, evaluate the mechanism by which this occurs and determine the dissemination to other organs. We will test strategies based on carrier cell homing and host conditioning to improve carrier cell access to the microenvironment and the efficiency of particle delivery. Finally, using a combination of biochemical assays and live-cell imaging we will investigate particle trafficking within carrier cells as a means of understanding and manipulating the mechanisms responsible for vector particle retention and release. The proposed strategy is based on our laboratory's observations, and adapts the established cancer gene therapy paradigm of using cellular carriers to target tumor tissue for virus particle delivery. Successful systemic delivery of lentivector particles by autologous cellular carriers directly to bone marrow stem cells will help overcome current impediments to gene therapy for hematopoietic and immune disorders. Without improved access to tissues and target cells, the therapeutic potential of gene therapy remains largely unfulfilled. The proposed development of a novel cellular approach to the delivery of genetic payload is therefore critically important not only for the intended correction of genetic disorders of the blood and immune system, but also as a tool for post injury repair in regenerative medicine.